Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Calcium Addition Potentially Reverses Lead <strong>and</strong> ManganeseInduced Enzymatic <strong>and</strong> Behavioral Alterations in RatsM. Ram Kumar, K. Praveen Kumar, V. Kavitha <strong>and</strong> G. Rajarami Reddy*Department <strong>of</strong> Zoology, Sri Venkateswara University, Tirupati – 517502, India*For Correspondence - gottipolu2002@yahoo.com1193AbstractChronic exposure to lead (Pb) ormanganese (Mn) is known to alter variety <strong>of</strong>neurological <strong>and</strong> behavioral functions. In thisstudy, we have examined the neuro-behavioralperturbations in rats exposed to both Pb <strong>and</strong> Mn,<strong>and</strong> the protective effect <strong>of</strong> calcium supplement.Rats (3 months old) were exposed to Pb (0.2%through drinking water) <strong>and</strong> Mn (intraperitoniallydaily at a concentration <strong>of</strong> 2.5 mg/kg body wt)for a period <strong>of</strong> 3 weeks.A separate batch <strong>of</strong> animals receivedcalcium (0.02%) in drinking water together withPb <strong>and</strong> exposed to Mn. Following exposures,the neurochemical alterations were assessed bydetermining the activity changes in synaptosomalacetyl cholinesterase (AChE) <strong>and</strong> mitochondrialATPases in different brain regions (cortex,hippocampus <strong>and</strong> cerebellum). Behavioral studiesincluded both cognitive (water maze tasks) <strong>and</strong>non-cognitive (open field, exploratory <strong>and</strong>locomotory tasks). Combined exposure to Pb <strong>and</strong>Mn resulted in behavioral dysfunctions (decreasein latency, swim distance, swim speed in watermaze task <strong>and</strong> decrease in open field behavior<strong>and</strong> locomotor activity). Similar to the changesobserved in motor <strong>and</strong> cognitive behavior,decrease in the activity <strong>of</strong> both AChE <strong>and</strong>ATPases were also observed in the brain regions<strong>of</strong> Pb+Mn exposed rats. However, the animalswhich received calcium together with Pb+Mnshowed reversal effect in behavioral as well asthe activity <strong>of</strong> the enzymes suggesting protectiverole <strong>of</strong> calcium supplementation against the Pb<strong>and</strong> Mn induced neurotoxicity. The results <strong>of</strong> thisstudy support our earlier findings on the protectiverole <strong>of</strong> calcium <strong>and</strong> zinc against Pb <strong>and</strong> Mninduced neurotoxicity.Keywords: Behavioral perturbations, Watermaze, Lead, Manganese, Locomotor activity,cholinergic system, Calcium protection.IntroductionThe nervous system is the primary targetfor the low levels <strong>of</strong> Pb-exposure <strong>and</strong> thedeveloping brain appears to be especiallyvulnerable to Pb/Mn-neurotoxicity (1-6). Pbexposureat very low doses produces seriousadverse effects on the central nervous system <strong>of</strong>children <strong>and</strong> infants, <strong>and</strong> these effects last forseveral years (7). Perinatal exposure to low levels<strong>of</strong> Pb has been shown to exert behavioral <strong>and</strong>neurochemical alterations in both suckling <strong>and</strong>adult rats (8). Pb is known to exert its neurotoxiceffects by competing with calcium for calciumreceptors coupled with second messengerfunctions (9) <strong>and</strong> in some cases, to inhibit theactions <strong>of</strong> Ca 2+ as a regulator <strong>of</strong> cell function (9).Calcium protection against lead <strong>and</strong> manganese toxicity
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1194Manganese (Mn) is an essential tracemetal that is present in all t<strong>issue</strong>s <strong>of</strong> mammals.This element can act both as a nutrient <strong>and</strong> atoxicant to the brain (10). The higher intake <strong>of</strong>Mn, either through the air or the diet, may resultin severe pathologies, particularly in the centralnervous system (CNS) leading to manganism, aParkinson-like disorder (11). The toxic effects <strong>of</strong>Mn have been widely studied using animalmodels, notably concerning the accumulation <strong>of</strong>Mn in t<strong>issue</strong>s <strong>and</strong> interactions with various otherelements (e.g., iron [Fe], zinc [Zn] copper [Cu])after subchronic or chronic administration (12).The adverse effects <strong>of</strong> Mn on thenervous system probably result from the failure<strong>of</strong> protective enzymes capable to detoxify criticalamounts <strong>of</strong> Mn or to alter its oxidation potential.A multifactor hypothesis is more likely, <strong>and</strong> couldinvolve iron-induced oxidative stress <strong>and</strong> thedirect interaction <strong>of</strong> Mn with dopaminergicterminal mitochondria, leading to selectivemitochondrial dysfunction <strong>and</strong> subsequentexcitotoxicity (13,14). In this study, we haveexamined the alterations in brain enzymes <strong>and</strong>behavioral perturbations in rats exposed to bothlead <strong>and</strong> manganese since both metals coexist inenvironment originating from different exposuresources. We have also studied the protectiveeffects <strong>of</strong> calcium against the alterations causedby Pb+Mn.Materials <strong>and</strong> MethodsProcurement <strong>and</strong> maintenance <strong>of</strong>experimental animals: Young albino rats(Wistar) <strong>of</strong> 3 months age were purchased fromIISc, Bangalore <strong>and</strong> maintained in the animalhouse <strong>of</strong> Dept. <strong>of</strong> Zoology, S.V. University. Theanimals were housed in clear plastic cages withhardwood bedding in a room maintained at 28 o ±2 o C <strong>and</strong> relative humidity 60 ± 10% with a 12hour light/day cycle. The animals were fed withst<strong>and</strong>ard pellet diet supplied by Sri VenkateswaraTraders, Bangalore <strong>and</strong> water ad libitum.Animal exposure to Pb <strong>and</strong> Mn: Animals wereexposed to 0.2% lead acetate (Sigma) by addingPb- acetate to de-ionized drinking water <strong>and</strong> Mn(2.5 mg/kg) (Sigma) intraperitonially daily for aperiod <strong>of</strong> 21 days. Control rats received onlydeionized water without Pb, intraperitonially saline(0.9%) injections daily up to 21 days.Calcium (Ca 2+ ) was supplemented as0.02% in 0.2% Pb- water. The behavioral tasksin control, Pb+Mn-exposed <strong>and</strong> calciumsupplemented 3 months old rats were observedin the open-field, water maze, exploratorybehavior chamber <strong>and</strong> locomotory activityrecorder. The brain regions (cerebral cortex,cerebellum <strong>and</strong> hippocampus) <strong>of</strong> control, Pb+Mnexposed,<strong>and</strong> Ca 2+ supplemented rats werecollected <strong>and</strong> used for biochemical assays.Behavioural StudiesOpen-Field Behavior: The open field test hasbeen widely used to assess emotional reactivity/anxiety. It provides measures <strong>of</strong> locomotoractivity. The horizontally directed activity (orlocomotion) is measured by the number <strong>of</strong> linecrossings, <strong>and</strong> vertically directed activity (orexploration) is measured by the frequency <strong>of</strong>rearings (15). The open-field behaviour <strong>of</strong> threemonths age rats was assessed in a wooden boxmeasuring 90 x 90 x 30 cm high. The floor <strong>of</strong> thearena was divided into 36 equal squares by blacklines. Immediately after a rat was placed in thecentre <strong>of</strong> the open field, the movement <strong>of</strong> theanimal was scored. The number <strong>of</strong> squarescrossed with all paws (crossings), the st<strong>and</strong>ingson the hind legs (rearings), placing the nose againstwall or floor (sniffing), wiping, licking, combingor scratching <strong>of</strong> any part <strong>of</strong> the body (grooming)were counted in all sessions. All the activitiesmeasured were combined together to assess themean total behaviour in each session. Testing wascarried out on five consecutive days in five minutesessions in control, Pb-exposed <strong>and</strong> supplementedanimals.Ram Kumar et al